WO2005040332A2 - Procede et bioreacteur pour la mise en culture et la stimulation de greffons cellulaires tridimensionnels, vitaux et mecaniquement resistants - Google Patents

Procede et bioreacteur pour la mise en culture et la stimulation de greffons cellulaires tridimensionnels, vitaux et mecaniquement resistants Download PDF

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Publication number
WO2005040332A2
WO2005040332A2 PCT/EP2004/011788 EP2004011788W WO2005040332A2 WO 2005040332 A2 WO2005040332 A2 WO 2005040332A2 EP 2004011788 W EP2004011788 W EP 2004011788W WO 2005040332 A2 WO2005040332 A2 WO 2005040332A2
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WIPO (PCT)
Prior art keywords
bioreactor
graft
chamber
reactor
mini
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PCT/EP2004/011788
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German (de)
English (en)
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WO2005040332A3 (fr
Inventor
Ronny Schulz
Augustinus Bader
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Universität Leipzig
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Application filed by Universität Leipzig filed Critical Universität Leipzig
Priority to CA002543374A priority Critical patent/CA2543374A1/fr
Priority to EP04790613A priority patent/EP1675939A2/fr
Priority to US10/576,618 priority patent/US20070026517A1/en
Priority to JP2006536026A priority patent/JP2007508830A/ja
Publication of WO2005040332A2 publication Critical patent/WO2005040332A2/fr
Publication of WO2005040332A3 publication Critical patent/WO2005040332A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/10—Perfusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli

Definitions

  • the invention relates to methods and an arrangement for GMP-compliant production of three-dimensional, vital and mechanically resistant cell cultures, preferably cartilage cell constructs, which can be cultured and stimulated in a novel manner in a closed mini-bioreactor simultaneously, sequentially or according to a time-controlled sequence.
  • These grafts grown in this way are available as tissue replacement material for the therapy of e.g. Connective and supporting tissue defects, direct joint trauma, rheumatism and degenerative joint diseases are available and can, e.g. in the case of knee osteoarthritis an alternative to conventional (operative) therapeutic approaches such as microfracturing or tapping.
  • Tissue engineering which is primarily concerned with the in vitro reproduction of autologous cell material, the body tries to grow functional cell and tissue replacement structures that can be inserted into the defective tissue in a transplant step.
  • cell cultures eg articular cartilage cells
  • chondrocytes e.g chondrocytes
  • the aim of these additive factors is, for example, to stimulate the special ability of cartilage cells to synthesize sufficient extracellular matrix components (EZM) to increase the mass ratio of 1% chondrocytes to 99% extracellular matrix components, as is the case in functional articular cartilage (Stockwell RA: The cell density of human articular and costal cartilage. J Anat. 1967; 101 (4): 753-763; Hamerman D, Schubert M: Diarthrodial joints, an essay. Amer J Med. 1962 33 : 555-590).
  • EMM extracellular matrix components
  • the dynamic cultivation scheme can guarantee a higher gas input and thus mechanically stimulates the cell aggregates depending on the selected medium perfusion flow, by means of a resulting shear force in the ⁇ Pa.
  • this passive system does not allow maximum gas exchange through the diffusion-permeable cover and the nutrient medium to the cell layer on the bottom.
  • a bioreactor device is also known from US Pat. No. 5,928,945, in which adherent cartilage cells are exposed to defined flows or shear forces in a growth chamber and consequently an increased type II synthesis of collagen was detected.
  • bioreactors which exert various mechanical loading processes on explants, cell samples or cell / polymer scaffolds.
  • their structure is based on the implementation of mechanical pressure stamps or the like. because they create uniaxial pressure on cartilage grafts to mimic the main form of stress on human cartilage. Many of these printing systems show great similarities in design.
  • Res Exp Med (Berl). 1993, 193 (3): 137-142 developed system consists of a titanium housing, which is covered on the inside with a polyethylene layer.
  • the test specimen with a maximum diameter of 10 mm can be placed in a container at the bottom of the chamber and covered with about 7 ml of artificial nutrient medium. Since the model does not have a perfusion system for the artificial culture medium, only batches of pressure can be generated with short cultivation times.
  • the load system which exerts corresponding pressure on the test specimen, consists of a porous pressure crucible that passes through the chamber closure and is moved either by simple weights or by an air cylinder with a pressure piston, which is attached above the chamber.
  • Lee DA, Bader DL Compressive strains at physiological frequencies influence the metabolism of chondrocytes seeded in agarose.
  • J Orthop Res. 1997; 15 (2): 181-188 published system which is actuated by a drive, is able to apply pressure to 24 test specimens simultaneously.
  • the drive is mounted on a frame that runs around the incubator and transfers the power down to the loading plate inside the sterile box.
  • the steel loading plate has 24 steel pins with a plexiglass notch of 11 mm in diameter.
  • the drive provides various loads that are made dependent on the degree of deformation.
  • This system is used for the cultivation of bovine chondrocyte / agarose scaffolds over a period of two days. Static and additional cyclic (0.3 to 3 Hz) loads with a maximum voltage amplitude of 15% are generated.
  • the disadvantage of a variety of pressure stimulation reactors is that the cell culture constructs during a Pressure load cannot be perfused with nutrient medium and therefore the effect of multiple cell irritation cannot be investigated. Furthermore, this lack of nutrients prevents an optimal metabolic exchange and the maximum synthesis of, for example, extracellular matrix components in cartilage cells.
  • Pressure and perfusion systems such as are described, for example, in US Pat. No. 6,060,306 and DE Patent 198 08 055, enable simultaneous multiple stimulation with parameters such as perfusion flow, the shear forces induced thereby and a uniaxial pressure load.
  • a disadvantage of reactors that enable pressure stimulation is above all that the pressure mediators, usually driven by servomotors or the like, usually plungers, pistons, etc. into the bioreactor room, where an autologous graft is preferably located, and then exert a defined pressure load on the cell construct.
  • the introduction of this pressure applicator into the sterile system makes the construction of closed pressure application reactors extremely difficult, so that these systems have an increased complexity.
  • the use of these (potentially non-sterile) systems is therefore only possible in basic research, since applications of these devices and methods in the medical field conflict with the guidelines of the existing pharmaceutical law.
  • the object of the invention is to provide a method and a bioreactor for the production of three-dimensional, vital and mechanically resistant cell cultures, in which cultivation and stimulation can take place in a close temporal connection or at the same time.
  • the bioreactor is intended to enable GMP-compliant graft cultivation under guaranteed sterile conditions.
  • the method and the bioreactor according to the invention combine the cultivation and stimulation of GMP-compliant, three-dimensional, vital and mechanically resistant cell cultures, preferably cartilage cell constructs, in one reactor.
  • the stimulation and cultivation can be carried out simultaneously, in succession or according to a time-controlled sequence.
  • These grafts bred in this way are available as Tissue replacement material for the treatment of, for example, connective and supporting tissue defects, direct joint trauma, rheumatism and degenerative joint diseases are available.
  • the essential feature of the method according to the invention and the bioreactor according to the invention is that there is a transplant in a closed reactor space which can be exposed to in vivo adaptive stimuli in several respects.
  • this closed bioreactor there is a magnetic, piston-like pressure temperature1 above the graft, which acts as a stress applicator on the cell culture.
  • the stamp is controlled contactlessly through the bioreactor room and thereby a directed uniaxial pressure stimulation on the tissue graft is brought about.
  • the contactless control of the mini actuator is carried out by externally arranged control magnets, whose aligned (electro) magnetic field causes a change in the position of the stamp in the bioreactor and results in organotypical dynamic or static pressure stimulation.
  • the method and the bioreactor have the advantage already mentioned that the cell cultures can also be stimulated during the cultivation. Above all, the cultivation or regeneration of connective and support tissue structures and functional tissue systems (cartilage, bones, etc.) is possible.
  • the apparatus enables the cultivation of cell transplants, in particular be synchronized, perfused and pressurized and are therefore characterized by an increased production of matrix components (e.g. cartilage cell cultures). Because of his
  • this device minimizes the number of operations and thus reduces the risk of infection
  • Design features of the bioreactor according to the invention ensure a closed bioreactor circuit and consequently strictly autologous cultivation or
  • Another area of application for the bioreactor is pharmaceutical active ingredient testing for the characterization of proliferation and differentiation-relevant substances or combinations of substances on transplants.
  • Fig. 7 Scheme of the technical device for construct perfusion and media mixing in Single-chamber bioreactor
  • Fig. 8 Scheme of the technical device for construct perfusion and media mixing in the bicameral bioreactor
  • Fig. 9 Fixation scheme of the graft in the bioreactor
  • Fig. 10 Magnet systems for controlling the mini-actuator.
  • Fig. 11 Stimulation scheme in the two-chamber reactor
  • FIG. 1 the use of the bioreactor for synchronous cultivation and stimulation of three-dimensional cell transplants is shown using the example of cartilage tissue transplantation.
  • the patient is the first (I) healthy cell material
  • the cells obtained are separated, counted and, depending on this, either sown using standard methods of tissue engineering in monolayer bottles (II) and reproduced strictly autologously or immediately sent to construct manufacture (III).
  • the cells are inserted into a three-dimensional graft structure made of biocompatible or resorbable carrier material (e.g. hydrogels, agaroses, collagens, hydroxylapatites, polymer complexes, etc.).
  • the suspended cells eg chondrocytes
  • the biogenic support structure eg agarose
  • a seed flask placed in a seed flask and hardened, for example, in a cylindrical graft shape (eg cartilage-agarose matrix).
  • a cylindrical graft shape eg cartilage-agarose matrix.
  • This seeding flask with the internal spatial cell graft is inserted into the bioreactor (IV), the graft is pressed out and positioned in the bioreactor.
  • the GMP-suitable cultivation and stimulation of this cell construct is then carried out either simultaneously, successively or in a time-controlled manner in the newly developed closed bioreactor apparatus (V).
  • the cell transplant can be brought to an increased differentiation and expression of organotypic markers with this multiple in vivo-like stimulation (stimuli such as shear force, perfusion, deformation, mechanical stress).
  • FIG. 2 illustrates an embodiment of the bioreactor system (with the two-chamber bioreactor) for autologous cultivation and multiple stimulation of cell transplants in a closed reactor structure using a GMP-compliant procedure.
  • the entire device for ensuring the optimal temperature, air humidity and composition is in a temperature-controlled manner and gas adjustable incubator.
  • the bioreactor 1 and the medium in the incubator and the other technical components are arranged outside the incubator for control purposes.
  • the bioreactor 1 itself and the components used therein are biologically and chemically inert and autoclavable.
  • the bioreactor body and the screw-on lid are made of non-magnetic (e.g. plastics) or weakly magnetic materials (e.g. vanadium-4 steel).
  • the culture medium is fed from the medium reservoir 2 through the hose system 4 with the 3-way valve 6 and the 4-way valve 7 into the bioreactor 1 by means of the circulation pump 5.
  • This culture medium can be enriched with autologous additive factors (for example growth factors, mediators, etc.) obtained from patient blood from the supplement reservoir 3.
  • the medium is fed to the bioreactor 1 and thus to the transplant 11 either in batch, fed-batch or in a continuous process.
  • the medium passes through the hose system 4 into the medium reservoir 2, which is equipped with measuring probes for checking the physicochemical parameters, such as pH, pC0 2 and p0 2 . If the medium is considered to be used up, it can be discharged via the hose system 4 into an external, sealed-off waste container. In both cases it is possible to use the valve device 7 to derive a sterile medium sample from the reactor circuit into a sampling section 8 for further analysis.
  • the graft 11 to be cultivated and stimulated lies in the medial position on the reactor floor.
  • a second, smaller chamber can be located below the graft 11.
  • This flow space is about the Hose system 4 is supplied with medium and can be filled with a highly porous but thin sintered material 16.
  • This lower chamber can be sealed off by a thin clear glass pane 17 and serve as a microscope opening for inverse microscopes.
  • This mini-actuator 14 which is designed as a magnetic stamp, acts as a pressure applicator without contact and is controlled by the control magnet or the coil 15.
  • FIG. 3 shows a possible embodiment of the bioreactor 1 consisting of a culture chamber which is used to implement the contactlessly controllable mini-actuator 14.
  • the bioreactor 1 which is designed as a single-chamber bioreactor, consists of a body and the bioreactor closure 21, which is additionally sealed by a squeeze ring 20.
  • Biosensors 9 are embedded in the cover construction, which serve for the on-line measurement of, for example, glucose and lactate concentrations, among other medium components.
  • In the reactor space there is a precisely fitted mini-actuator 14 above the graft 11, which rests on a special reactor floor with a clear glass pane 17 inserted.
  • a sampling section 8 is integrated into at least one of the outlets 19 via a 3-way valve 6.
  • FIG. 4 outlines a further embodiment of a bioreactor 1 consisting of two chamber spaces, the upper one containing the pressure stamp 14 and the lower one serving for the flow below the transplant 11.
  • This embodiment does not differ in function, property and requirement of components 1, 6, 8, 9, 14, 19-21 from the bioreactor 1 described in Example 3.
  • In the apparatus there are at least one inlet and outlet 19 in the upper one and lower reactor chamber let in to achieve a valve-controlled flow to the respective chamber and the graft 11.
  • the dimension of the lower chamber is such that its diameter is less than that of the graft 11.
  • This chamber accommodates a flat, precisely fitting disc made of porous sintered material 16, so that inverse microscopy through the closing glass disc 17 and the membrane 18 to the transplant 11 can take place without being impaired.
  • This disk of sintered material 16 in the lower reactor chamber fulfills another important function in the present apparatus. It prevents an undesired pressing of the gel-like cell construct 11 into the chamber space when the graft 11 is mechanically loaded by the pressure stamp 14.
  • the use of a fluid-permeable membrane 18 between the sintered material 16 and the graft 11 is provided in order to prevent the carrier material from being mixed with the sintered material 16.
  • FIGS. 5 show the structure, the geometry and the differing shapes of the mini-actuator 14, which slides vertically in the reactor space (here exemplarily in the two-chamber model) and transmits axial compressive forces to the graft 11 lying on the reactor floor.
  • This magnetic pressure applicator 14 is contactless according to the invention (see FIG. 5a) by externally arranged ones
  • FIG. 5b shows the characteristic structure of this pressure unit 14. It has an extremely powerful permanent magnet 22, preferably made of a neodymium-iron-boron compound even with the slightest magnetic and electromagnetic fields moved in the respective field direction.
  • This permanent magnet 22 is encapsulated in a lacquered or galvanized form in a biologically inert plastic — the enveloping body 23.
  • This, preferably cylindrical, envelope body 23 slides with its precisely fitting outer diameter with little friction and exactly vertically in the bioreactor cylinder.
  • other organotypical negative shapes can also be embossed as a stamp surface 24 on the underside of the plastic enveloping body 23 in order to reproduce in-vivo adaptive positive shapes (including curvatures, arches, etc.).
  • FIG. 5 c shows a further exemplary embodiment of the mini actuator 14, which is likewise constructed from a strong permanent magnet 22 and an enveloping body 23 with an individual stamp surface 24.
  • this model has so-called flow channels 33 at the edge of its envelope 23. This enables media to flow around the mini-actuator 14 in the bioreactor space and lower actuating forces are required to overcome the media resistance.
  • the enveloping body 23 must have at least 3 guide lugs with a precisely fitting outer diameter D2 in order to ensure a planar positioning of the entire mini-actuator 14 on the transplant 11.
  • FIG. 5d shows a modified pressure stamp 14 based on FIG. 5b, which has an extension web 34 to create a spatial distance between the permanent magnet 22 and the cell culture construct 11.
  • the reason for this spacing of the permanent magnet 22 in the upper cylinder head from the graft 11 is the minimization any field influences on cell cultures 11.
  • FIG. 5e shows a mini-actuator 14 based on FIG. 5d, which has at least 3 flow channels 33 and 3 guide lugs with an outer diameter D2.
  • FIG. 6 shows the method and the device for producing and sowing three-dimensional, preferably cylindrical, cell matrix constructs.
  • FIG. 6a cell matrix seed
  • increased see FIG. 1, II
  • freshly isolated see FIG. 1, III
  • prepared cells 12 are mixed with the biogenic carrier structure 13, suspended until homogeneous and with the target volume of the cell matrix in injected the plunger 25.
  • the precisely fitting seed piston 25 corresponds in its inner diameter D1 to the future outer diameter of the graft 11 and in its outer diameter D2 to the inner diameter of the bioreactor 1.
  • FIG. 6b stamp insert shows the stamp insert 26 in the seed piston 25.
  • stamp insert shows the stamp insert 26 in the seed piston 25.
  • the precisely fitting planar punch 26 with the outer diameter D1 is inserted into the hollow piston cylinder on the flat sliding plate 27.
  • the underside of this stamp 26 can be organotypically analogous to the stamp surface 24 of the mini actuator 14 Structures.
  • FIG. 6c shows the attachment of the stamp 26 on the transplant 11 in the seed piston 25.
  • the stamp 26 is placed on the cell structure with a slight pressure in order to counteract meniscus formation or curvature of the matrix top of the transplant 11, e.g. to get a cylindrical graft shape. If an in vivo adaptive surface is to be imprinted on the graft 11, this stamp attachment 26 must take place during the curing or polymerization phase.
  • the attached stamp 26 is lifted after the graft 11 has been shaped and the, preferably hydrophobic, sliding plate 27 located at the bottom of the seed piston 25 is removed.
  • the, preferably hydrophobic, sliding plate 27 located at the bottom of the seed piston 25 is removed.
  • an inert film or an inert polymer fleece is used to line the surfaces.
  • FIG. 6e shows the sowing of a cylindrical construct using the example of the two-chamber bioreactor.
  • the precisely fitting seed piston 25 is implemented in the bioreactor 1, then the cell construct 11 is introduced medially into the prepared reactor by means of a pressure stamp 26 and the sowing device is removed from the bioreactor 1.
  • This prepared bioreactor 1 contains the porous sintered material 16 and optionally a diffusion-permeable membrane 18.
  • inlets and outlets with an integrated Luer coupling 19 open into the bioreactor 1. These can differ both in their position and position for flow optimization, i.e. also e.g. Enter the bioreactor body 1 tangentially.
  • the number of inlets and outlets 19 leading into bioreactor 1 is at least two.
  • a sampling section 8 can be installed at each flowing Luer connector 19, e.g. a 3-way valve 6 at each flowing Luer connector 19, e.g. a 3-way valve 6 a sampling section 8 can be installed.
  • the medium diffuses primarily into the upper and lateral edge areas of e.g. cylindrical tissue graft 11 and supplies the cell culture and others. with nutrients and at the same time removes metabolic end products from the carrier matrix.
  • FIG. 7b shows a continuous supply of nutrient medium from the medium reservoir 2 with an optional one behind it
  • Supplement reservoir 3 (not shown) by means of a meterable circulation pump 5 through the hose system 4 into at least one inlet 19 of the bioreactor 1.
  • the medium used can, as shown here, in The circuit remains in that it enters the medium reservoir 2 and from there is used again for the continuous perfusion of the graft 11. Otherwise it can be completely removed from the circuit.
  • the graft 11 is then cultivated in a batch or fed-batch process.
  • a targeted, continuous supply of culture medium into the reactor space can significantly improve the inflow and throughflow of the graft 11 in comparison to the static scheme of FIG. 7a. Due to the induced perfusion, deeper construct regions are flushed out with medium. As a result, the metabolism is optimized, which can lead to increased cell differentiation. Furthermore, this design of the flow of the construct exerts a shear force stimulation on the embedded cells.
  • FIGS. 8 show a bicameral bioreactor which allows an optimized flow, diffusion and perfusion of the graft and thus helps to improve the quality of the tissue replacement.
  • FIG. 8a A variant with static cultivation and diffusion is shown in FIG. 8a.
  • the inlets and outlets 19 opening into the bioreactor 1 are at least two, at least one each having to open into the lower and upper reactor chambers.
  • the two inlets and outlets 19 per chamber listed here can differ in terms of their position, position and thickness for flow optimization.
  • the sampling section 8 can be installed on each outflow-oriented Luer connector 19 of both chambers, for example with a 3-way valve 6 or the like.
  • the chamber newly created in this construction below the graft 11 during the static cultivation phase leads to a diffusion of the culture medium from the porous sintered material into the regions of the support structure near the bottom, resulting in an improved metabolism in the entire graft 11 results.
  • FIG. 8b shows the continuous supply of nutrient medium from the medium reservoir 2 with an optional supplementary reservoir 3 (not shown) arranged behind it by means of a meterable circulation pump 5 or the like. through the hose system 4 into at least one inlet 19 of the lower and upper chamber of the bioreactor 1. The medium is discharged through at least one outlet 19 per chamber into the hose system 4, at which at least one point via a 3-way valve 6 uncoupled sampling section 8 can be integrated.
  • the used medium can remain in the circuit by entering the medium reservoir 2 and from there being used again for the continuous perfusion of the transplant 11. Otherwise it can be completely removed from the circuit.
  • the graft 11 is cultivated in a batch or fed-batch process.
  • the integration of a second chamber according to the invention, here below the transplant 11, shows its positive properties, above all when the biological construct is subjected to a targeted flow. Accordingly, if the 3- Directional valve 6 of the media flow from the medium reservoir 2 into the lower chamber, an induced upward perfusion of the graft 11 takes place with the lower drain closed, since the medium can only leave the reactor space through the upper drain.
  • FIG 9 shows the fixation scheme of the graft 11 in the bioreactor 1 either in the single-chamber or two-chamber version.
  • FIG. 9a shows the graft 11 to be stimulated, which is fixed medially above the clear glass 17 in the single-chamber bioreactor 1.
  • FIG. 9b shows the use of at least 3 of these fixation walls 28 in the two-chamber bioreactor in order to achieve a horizontal fixation of the graft 11 in the various flow conditions and to enable ideal vertical perfusion and mechanical pressurization.
  • FIGS. 10 (shown on the single-chamber bioreactor) represent characteristic devices and apparatus arrangements for the contactlessly controllable stimulation method of the mini-actuator 14 on the transplant 11.
  • FIG. 10a magnetic control effect of magnetic attraction
  • FIG. 10a shows the characteristic device and the principle for contactless control of the magnetic
  • Permanent magnets in the mini actuator 14 take place according to the predominant magnetic field direction, which is generated by externally arranged control magnets 15.
  • Control magnet 15 e.g. is at least one permanent magnet or at least one coil, a defined one
  • Mini actuator 14 triggers.
  • the control magnet 15 shows the principle of magnetic attraction.
  • the magnetic repulsion represents the second magnetic control effect between the magnetic control system 15 and the mini actuator 14.
  • FIG. 10c (control of the mini-actuator 14 by means of a control magnet guide plate) represents an embodiment of a permanent magnetic control system.
  • a control magnet guide plate represents an embodiment of a permanent magnetic control system.
  • an arrangement of several permanent magnets 32 of different sizes, polarities and thus field strength and direction works on a linearly controlled guide plate 31 which is positioned here as an example above the prototype reactor.
  • a linear rail 29 drives a guide rail 30 with the permanent magnets 32 inserted in the magnet holder 31. This mobile phase of the magnet system makes movement of the bioreactor 1 unnecessary.
  • control system in FIG. 10d (control of the mini actuator 14 by means of rotating permanent magnets) is based also on a control of the magnetic pressure stamp 14 by means of a permanent magnet arrangement on a rotating disc.
  • an actuator 29 drives a magnet holder 31 with fitted permanent magnets 32 of alternating polarities in a rotating manner.
  • This rotating magnet holder can be occupied, for example, with four alternately polarized magnets 32 and, as a result, causes two complete pressurizations on the graft 11 during a full revolution.
  • the combination of this magnet occupation of the rotating disc with the rotational speed of the servomotor 29 results in a higher frequency magnetic field change and consequently a highly dynamic one Stimulation scheme for the graft 11.
  • the front view illustrates both magnetic effects of the rotation system using two bioreactors 1.
  • the embodiment of this device is suitable for a large number of bioreactors 1, as long as they can be placed exactly above or below the control magnet center.
  • FIG. 10e represents a magnet device based on a coil arrangement.
  • This magnet coil system works with an induction coil 35, which is fixed here above the bioreactor 1, producing a defined electromagnetic field which is distributed over the supplied electrical power can be regulated continuously and thus enables any positions of the mini-actuator 14 in the bioreactor body. By reversing the polarity of the current direction, the existing field direction and the electromagnetic effect are reversed.
  • the iron core coil 35 used builds up its electrical field perpendicular to the coil winding and acts on it static permanent magnets of the mini actuator 14 attracting and repelling one.
  • An automated station of this system consists of a powerful coil 35 with low heat development with a connected adjustable transformer, the performance of which is monitored by means of a multimeter.
  • a microcontroller ensures the activation of a relay, which switches the current in the desired direction, the desired effect of an intermittent pressure application on the cell construct.
  • FIG. 11 shows the entire stimulation scheme of the novel GMP-capable bioreactor 1.
  • the mechanical pressure stimulation, the perfusion and the shear force-induced flow in the three-dimensional graft 11 run in parallel.
  • the cell construct 11 is stimulated only via a directed flow of media, which leads to a perfusion of the construct with exerting a shear force on the
  • FIG. 11 b perfusion stimulation and stamp attachment shows the second step of multiple stimulation of tissue replacement materials 11 in the bioreactor 1. First, as in this example, the flow conditions are modified.
  • the nutrient medium flow is directed only into the lower reactor chamber, from where it perfuses through the graft 11, brings about the mass transfer and can leave the upper reactor chamber via a drain.
  • the control magnet system here an iron core coil 35 with low power induction
  • the magnetic mini-actuator 14 is attached to the cylindrical tissue replacement 11, for example.
  • This stamp attachment with 0% construct deformation marks a reversal point of the mini-actuator 14 at one dynamic high-frequency deformation of the cell matrix 11.
  • FIG. 11c perfusion stimulation and mechanical stress
  • the stamping device After the pressure loading protocol has been processed, the stamping device is returned to its initial state, the cell culture is perfused, for example, continuously and the transplant 11 is removed, for example if the extracellular matrix is sufficiently synthesized.

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  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

L'objectif de l'invention est d'offrir un procédé et un bioréacteur pour la réalisation de cultures cellulaires tridimensionnelles, vitales et mécaniquement résistantes, procédé selon lequel la mise en culture et la stimulation peuvent se faire dans un contexte temporel étroit ou de façon simultanée. Le bioréacteur doit permettre une mise en culture de greffons selon les normes GMP, dans des conditions stériles garanties. Le bioréacteur (1) selon l'invention est constitué d'un corps de base, qui est assemblé avec un dispositif de fermeture de réacteur (21) de façon étanche à la pression et stérile et forme au moins une chambre de réacteur dans laquelle se trouvent une surface de support destinée à un greffon (1) et un mini-actionneur (14). En outre, le bioréacteur (1) est pourvu d'au moins deux raccords pour tuyaux flexibles (19) qui servent à l'amenée et à l'évacuation du milieu de culture ou à l'apport de gaz. L'invention permet la réalisation selon les normes GMP de cultures cellulaires tridimensionnelles vitales et mécaniquement résistantes, de préférence de constructions cellulaires de cartilage, la mise en culture et la simulation pouvant se faire dans un mini-bioréacteur fermé, de façon simultanée, séquentielle ou selon un déroulement temporellement commandé. Les greffons résultant de cette culture peuvent être utilisés comme matériaux de remplacement de tissus pour le traitement, par exemple, de défauts du tissu conjonctif ou du stroma, de traumatismes articulaires directs, des rhumatismes et des maladies articulaires dégénératives, et ils peuvent, par exemple en cas d'arthrose de l'articulation du genou, être utilisés en tant qu'autres solutions de traitement pour remplacer les traitements (par opération) habituels tels que la microfracturation ou le perçage.
PCT/EP2004/011788 2003-10-21 2004-10-19 Procede et bioreacteur pour la mise en culture et la stimulation de greffons cellulaires tridimensionnels, vitaux et mecaniquement resistants WO2005040332A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA002543374A CA2543374A1 (fr) 2003-10-21 2004-10-19 Procede et bioreacteur pour la mise en culture et la stimulation de greffons cellulaires tridimensionnels, vitaux et mecaniquement resistants
EP04790613A EP1675939A2 (fr) 2003-10-21 2004-10-19 Procede et bioreacteur pour la mise en culture et la stimulation de greffons cellulaires tridimensionnels, vitaux et mecaniquement resistants
US10/576,618 US20070026517A1 (en) 2004-10-19 2004-10-19 Method and bioreactor for the cultivation and stimulation of three-dimensional, vitally and mechanically reistant cell transplants
JP2006536026A JP2007508830A (ja) 2003-10-21 2004-10-19 三次元、生体及び機械的抵抗性を有する細胞移植体の培養と刺激のための方法とバイオ反応装置

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DE10349484.7 2003-10-21
DE2003149484 DE10349484A1 (de) 2003-10-21 2003-10-21 Verfahren und Bioreaktor zum Kultivieren und Stimulieren von dreidimensionalen, vitalen und mechanisch widerstandsfähigen Zelltransplantaten

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WO2005040332A2 true WO2005040332A2 (fr) 2005-05-06
WO2005040332A3 WO2005040332A3 (fr) 2005-06-30

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JP (1) JP2007508830A (fr)
CN (1) CN1898375A (fr)
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DE (1) DE10349484A1 (fr)
RU (1) RU2370534C2 (fr)
WO (1) WO2005040332A2 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009047045A2 (fr) * 2007-10-09 2009-04-16 Politecnico Di Milano Bioréacteur pour la génération et la stimulation mécanique complexe d'un tissu biologique synthétisé par génie génétique
WO2009118140A2 (fr) * 2008-03-25 2009-10-01 Novatissue Gmbh Bioréacteur à perfusion pour produire des tissus humains ou animaux
WO2009118141A2 (fr) * 2008-03-25 2009-10-01 Novatissue Gmbh Bioréacteur à perfusion pour fabrication et/ou culture d'un vaisseau sanguin humain ou animal et/ou d'un tissu humain ou animal
WO2009141163A2 (fr) * 2008-05-23 2009-11-26 Greiner Bio - One Gmbh Bioréacteur et procédé pour culture de cellules et de tissus
EP2184347A2 (fr) 2008-11-06 2010-05-12 Georg N. Duda Nouveaux composants et nouveau système de bioréacteurs pour la culture et la stimulation mécanique de matériau biologique
ITMI20090388A1 (it) * 2009-03-13 2010-09-14 Istituto Ortopedico Galeazzi S P A Bioreattore per la coltura di cellule o tessuti biologici, relativo metodo e sis tema multistazione
DE102009050498A1 (de) 2009-10-23 2011-04-28 Universität Leipzig Perfundierbarer Bioreaktor zur Herstellung von menschlichen und tierischen Geweben
WO2013117193A1 (fr) * 2012-02-09 2013-08-15 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V. Cellule de stimulation et procédé de stimulation de cellules ou tissus in vitro
ITUB20160272A1 (it) * 2016-01-22 2017-07-22 Univ Degli Studi Di Palermo Bioreattore a perfusione autosufficiente monouso per crescite cellulari 3D

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* Cited by examiner, † Cited by third party
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DE102005049905A1 (de) * 2005-10-17 2007-04-19 Medizinische Hochschule Hannover Verfahren und Vorrichtung zur Herstellung eines Gelenkersatzes
JP5433129B2 (ja) 2006-07-10 2014-03-05 パーパス株式会社 細胞又は組織の培養方法
JP4981374B2 (ja) 2006-07-10 2012-07-18 パーパス株式会社 細胞又は組織の培養装置及び培養方法
DE102008050424B4 (de) 2008-10-08 2010-11-25 Universität Leipzig Verfahren und Vorrichtung zur homogenen Verteilung einer zellulären Suspension in porösem Trägermaterial für die Herstellung von vitalem biologischem Ersatzgewebe
BR112012015653B1 (pt) 2009-12-29 2023-09-26 W.R. Grace & Co. -Conn. Composição que compreende um material de sílica particulado poroso e película transparente que compreende a mesma
DE102010026369B4 (de) 2010-07-07 2015-07-30 Zellwerk Gmbh Verfahren und Druck-Drehbett-Bioreaktor zur dynamischen Expansion und/oder Differenzierung von Stammzellen oder primären Zellen humanen und tierischen Ursprungs
KR101313769B1 (ko) 2011-02-28 2013-10-01 주식회사 셀루메드 동종 연조직 이식체 배양 장치
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WO2014132101A2 (fr) * 2012-10-18 2014-09-04 Yongxin Zhang Système de bioréacteur et procédés de culture cellulaire alternant entre un état statique et un état dynamique
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RU190863U1 (ru) * 2019-02-15 2019-07-15 Федеральное государственное бюджетное учреждение "Российский ордена Трудового Красного Знамени научно-исследовательский институт травматологии и ортопедии имени Р.Р. Вредена" Министерства здравоохранения Российской Федерации (ФГБУ "РНИИТО им. Р.Р. Вредена" Минздрава России) Устройство для совмещения клеточной культуры и биодеградируемого носителя
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AT523906B1 (de) * 2020-10-02 2022-01-15 Inncellys Gmbh Inkubationskammer

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996039992A1 (fr) * 1995-06-07 1996-12-19 Advanced Tissue Sciences, Inc. Appareil et procede pour steriliser, ensemencer, cultiver, stocker, expedier et tester des tissus cartilagineux de remplacement produits par assemblage
DE19808055A1 (de) * 1998-02-27 1999-09-02 Adamietz Verfahren und Apparatur zur Herstellung von dreidimensionalen Gewebezellkulturen
US20010043918A1 (en) * 2000-05-05 2001-11-22 Masini Michael A. In vitro mechanical loading of musculoskeletal tissues
WO2002048317A2 (fr) * 2000-12-12 2002-06-20 Bauer Joerg Procede et dispositif de fabrication de tissu biologique dans une chambre de croissance
WO2003029398A1 (fr) * 2001-08-30 2003-04-10 Takagi Industrial Co., Ltd. Incubateur pour cellules et structures

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4306661C2 (de) * 1993-03-03 1995-04-20 Michael Dipl Biol Sittinger Verfahren zum Herstellen eines Implantates aus Zellkulturen
DE19962456A1 (de) * 1999-12-22 2001-07-12 Biotechnologie Ges Mittelhesse Verfahren und Apparatur zur Herstellung, Untersuchung und mechanischen Stimulation dreidimensionaler Gewebezellkulturen
DE10201259A1 (de) * 2002-01-15 2003-08-28 Augustinus Bader Vorrichtung zum Züchten oder Kultivieren von Zellen in einem dosenartigen Behälter

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996039992A1 (fr) * 1995-06-07 1996-12-19 Advanced Tissue Sciences, Inc. Appareil et procede pour steriliser, ensemencer, cultiver, stocker, expedier et tester des tissus cartilagineux de remplacement produits par assemblage
DE19808055A1 (de) * 1998-02-27 1999-09-02 Adamietz Verfahren und Apparatur zur Herstellung von dreidimensionalen Gewebezellkulturen
US20010043918A1 (en) * 2000-05-05 2001-11-22 Masini Michael A. In vitro mechanical loading of musculoskeletal tissues
WO2002048317A2 (fr) * 2000-12-12 2002-06-20 Bauer Joerg Procede et dispositif de fabrication de tissu biologique dans une chambre de croissance
WO2003029398A1 (fr) * 2001-08-30 2003-04-10 Takagi Industrial Co., Ltd. Incubateur pour cellules et structures

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009047045A2 (fr) * 2007-10-09 2009-04-16 Politecnico Di Milano Bioréacteur pour la génération et la stimulation mécanique complexe d'un tissu biologique synthétisé par génie génétique
WO2009047045A3 (fr) * 2007-10-09 2010-01-14 Politecnico Di Milano Bioréacteur pour la génération et la stimulation mécanique complexe d'un tissu biologique synthétisé par génie génétique
WO2009118141A3 (fr) * 2008-03-25 2011-12-29 Novatissue Gmbh Bioréacteur à perfusion pour fabrication et/ou culture d'un vaisseau sanguin humain ou animal et/ou d'un tissu humain ou animal
WO2009118140A2 (fr) * 2008-03-25 2009-10-01 Novatissue Gmbh Bioréacteur à perfusion pour produire des tissus humains ou animaux
WO2009118141A2 (fr) * 2008-03-25 2009-10-01 Novatissue Gmbh Bioréacteur à perfusion pour fabrication et/ou culture d'un vaisseau sanguin humain ou animal et/ou d'un tissu humain ou animal
WO2009118140A3 (fr) * 2008-03-25 2012-01-05 Novatissue Gmbh Bioréacteur à perfusion pour produire des tissus humains ou animaux
WO2009141163A2 (fr) * 2008-05-23 2009-11-26 Greiner Bio - One Gmbh Bioréacteur et procédé pour culture de cellules et de tissus
WO2009141163A3 (fr) * 2008-05-23 2012-01-12 Greiner Bio - One Gmbh Bioréacteur et procédé pour culture de cellules et de tissus
EP2184347A2 (fr) 2008-11-06 2010-05-12 Georg N. Duda Nouveaux composants et nouveau système de bioréacteurs pour la culture et la stimulation mécanique de matériau biologique
ITMI20090388A1 (it) * 2009-03-13 2010-09-14 Istituto Ortopedico Galeazzi S P A Bioreattore per la coltura di cellule o tessuti biologici, relativo metodo e sis tema multistazione
DE102009050498A1 (de) 2009-10-23 2011-04-28 Universität Leipzig Perfundierbarer Bioreaktor zur Herstellung von menschlichen und tierischen Geweben
WO2013117193A1 (fr) * 2012-02-09 2013-08-15 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V. Cellule de stimulation et procédé de stimulation de cellules ou tissus in vitro
ITUB20160272A1 (it) * 2016-01-22 2017-07-22 Univ Degli Studi Di Palermo Bioreattore a perfusione autosufficiente monouso per crescite cellulari 3D

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WO2005040332A3 (fr) 2005-06-30
EP1675939A2 (fr) 2006-07-05
JP2007508830A (ja) 2007-04-12
RU2370534C2 (ru) 2009-10-20
CN1898375A (zh) 2007-01-17
RU2006117360A (ru) 2007-11-27
CA2543374A1 (fr) 2005-05-06
DE10349484A1 (de) 2005-05-25

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